Neisseria gonorrhoeae (Ng) is the causative agent of the sexually transmitted infection, gonorrhea. Gonorrhea causes over 100 million new cases worldwide annually and adversely affects the reproductive health of women. Ng has become resistant to almost every antibiotic that has been used and has now achieved superbug status. Vaccines against Ng are urgently needed. Complement (C) is a critical arm of innate immune defenses against Ng. Gonorrhea is characterized by an influx of polymorphonuclear leukocytes (PMNs) into the genital tract, which in concert with Ab and C may facilitate clearance of infection. Vaccine development against Ng has been hindered by lack of knowledge of the correlates of protection. Our vaccine candidate is a peptide mimic (mimitope) of a Ng lipooligosaccharide (LOS) epitope that elicits bactericidal and opsonophagocytic Ab in mice. In Specific Aim 1, we will elucidate the roles of PMNs, C and C receptors in vaccine Ab efficacy to define the correlates of protection in vivo. Vaccine Ab will be evaluated in mice that lack either PMNs, C3 (opsonin) or C5a (chemotaxin) receptor. Ng binds the C inhibitors, factor H (fH) and C4b- binding protein (C4BP) in a human-specific manner. Binding of human C inhibitors blunts the bactericidal activity of vaccine Ab. We have developed transgenic mice that express both human fH and C4BP, which will be used to evaluate the efficacy of vaccine Ab in vivo and simulate conditions in humans where Ab must surmount human C inhibitors. Ng LOS plays a central role in the pathogenesis of gonorrhea. The ability of Ng to alter its LOS glycan extensions through phase variation contributes to its versatility in immune evasion. We have constructed eight Ng mutants where LOS glycan expression has been 'genotypically fixed'. In Specific Aim 2a the ability of each of these mutants to infect mice will be examined, which will elucidate how defined LOS structures driven by fixed genotypes contribute to bacterial fitness in vivo. In light of our data which show that altering LOS glycans modulates the bactericidal activity of vaccine anti-LOS Ab, we will study the efficacy of the vaccine Ab against each 'fit' LOS genotype in Specific Aim 2b. Studies in Aim 2 will correlate LOS geno/phenotypes with fitness and enable us to identify LOS structures that could supplement our current vaccine candidate. Individuals who possess Ab directed against an Ng conserved and ubiquitous outer membrane protein called reduction modifiable protein (Rmp) show impaired bactericidal activity against Ng and are at a higher risk of contracting gonorrhea. In Specific Aim 3 we will determine the molecular basis whereby Abs directed against Rmp block C-dependent Ab killing of Ng and negatively impact the efficacy of vaccine Ab. Interestingly, anti-Rmp Ab does not block killing of N. meningitidis (Nm). Allelic replacement of rmp across the two species will be performed to define whether subtle sequence differences between Ng and Nm Rmp contribute to differences in blocking across the two pathogens. Corresponding Ng and Nm PorB molecules will also be exchanged across the two species to assess co-dependence of species specific PorB on the blocking effect. Completion of the proposed studies will represent an important step forward in the development of novel vaccines against gonorrhea.